Kex2蛋白酶K291突变体性质和动力学研究

Properties and Kinetics of Kex2 Protease Mutants K291

利用毕赤酵母GS115表达系统,成功表达并纯化获得了两种Kex2蛋白酶突变体Kex2-K291L和Kex2-K291H。对比研究了天然Kex2蛋白酶和这两种突变体的酶学性质和稳定性。实验结果表明,与天然Kex2蛋白酶相比,Kex2-K291H和Kex2-K291L两种突变体的稳定性得到了明显的改善。两种突变体的最适pH和最适温度与天然Kex2蛋白酶保持一致,均为pH 9.0和37℃,两种突变体的温度稳定性也与天然Kex2蛋白酶基本一致。与天然Kex2蛋白酶相比,突变体Kex2-K291H的pH值稳定性范围从5.0~6.0扩大至5.0~7.0。蛋白酶促反应动力学研究表明,突变体Kex2-K291H和Kex2-K291L的K_(cat)/K_(m)值分别为天然Kex2蛋白酶的1.85倍和2.05倍。突变体Kex2-K291H和Kex2-K291L在改善天然Kex2蛋白酶自降解问题的同时,提高了pH稳定性和催化效率。

Two kinds of Kex2 protease mutants,Kex2-K291L and Kex2-K291H were successfully expressed in Pichia pastoris,which were induced by methanol and purified with anion exchange chromatography(Q-FF).Finally,the enzymatic characteristics of these Kex2 proteases were characterized.Compared with the wild-type Kex2,the degradation of the two mutants Kex2-K291H and Kex2-K291L was significantly improved.The wild-type Kex2 protease was degraded during the purification,and a non-single band appeared,while the mutants were not degraded during the purification,and it was still a single band.The optimum pH and temperature of these two mutants were the same as those of the wild-type Kex2.Their optimal pH and temperature were pH 9.0 and 37℃,respectively.Compared with the wild-type Kex2 protease,the pH stability range of the mutant Kex2-K291H was expanded,from the range of pH 5.0 to 6.0 to the range of pH 5.0 to 7.0.Compared with the wild-type Kex2,Kex2-K291H was more stable at the temperature range of 4℃to 37℃.Enzymatic reaction kinetics studies showed that the K_(cat)/K_(m) values of mutant Kex2-K291H and Kex2-K291L were 1.85 and 2.05 fold higher than that of the wild-type Kex2 protease,respectively.